Cell Culture Device and Cell Culture Method

The present invention relates to a cell culture device and a cell culture method using the cell culture device.

Conventionally, in the field of medicine or drug development, cells are cultured and analyzed in cell culture devices to elucidate structure and function of various types of cells, tissues, organs in vivo. As analysis methods using such cell culture devices, for example, a method for high-throughput analysis of cellular functions at the single-cell level by forming an array of many minute cell culture sections using microfabrication technique based on semiconductor processing technique and culturing a single cell in each cell culture section, a method of analyzing cell-to-cell communication by co-culturing various types of cells in minute cell culture sections formed by microfabrication technique, and further a method of analyzing pharmacokinetics, etc. by using tissues or organs reconstructed in minute cell culture sections are known.

As cell culture device used in such analysis method, for example, a cell culture device wherein each well bottom surface of microwell array is constituted of self-supported membrane made by SiN by semiconductor processing technique, and multiple micro through-holes having a diameter of about 3 μm are formed on the SiN membrane is proposed (see non Patent Literature 1). By culturing multiple astrocytes on the back surface of the SiN membrane, and a single neuron on the surface of SiN membrane in each well, cell-to-cell communication through micro-through holes became possible, and it has been succeeded to maintain for a long period the physiological activity of a single neuron.

However, when seeding directly cells to microwell array using micropipette, the probability of introducing one cell into one well was low, and it was very difficult. In case of making the size of the well as small as about the cell size, since physically two or more cells can hardly be introduced in one well, and the success rate would be relatively high. However, in case of culturing adherent cells that require a large culture surface area such as neuron, the size of the well being necessary large, and it was difficult to introduce one cell in one well, and the success was about a few %.

Further, a double layer micro-flow channel device separated by porous membrane made by semiconductor processing technique is proposed (see Patent Literature 1). In the first micro-flow channel, a retinal layer is formed by culturing retinal pigment epithelium cells, and in the second micro-flow channel, a blood vessel layer is formed by culturing vascular endothelial cells and fibroblast, and thereby a model of blood-retinal barrier consisting of two layers is formed.

However, by applying such double layer type micro flow channel device separated by a porous membrane, to form tissues having three or more layers, it was necessary to provide two or more porous membranes in the micro flow channel. Further, such device is made by sticking the upper and lower micro flow channels produced by semiconductor processing technique and the porous membrane consisting of plastic material such as polyethylene terephthalate (PET), and when the number of layer increases, the positioning accuracy when sticking worsens. Further, the distance between layers was hard to control, and for example, even if the cells of the first layer and the second layer could be arranged in the near distance separated by the porous membrane, it was difficult to get closer the cells of the second layer and the third layer to an appropriate distance.

[Patent Literature 1] Japanese Patent Laid-Open Publication No. 2020-188723

[Non-Patent Literature 1] Yusuke Zenmyo, Isamu Morisako, Takashi Yasuda; Long-term Culture of a Single Neuron using a Microwell with a Porous SiN Membrane, Article of The Institute of Electrical Engineers of Japan E, Vol. 138, No. 7, pp. 327-328 (2018)

The object of the present invention is to provide a new cell culture device, and a culture method using such cell culture device.

The present inventors found a new cell culture device useful for analysis of function at a single cell level, or analysis of cell-to-cell communication, etc. and a culture method using the cell culture device. The present invention has been thus completed.

Specifically, the present invention relates to the following.

[1] A cell culture device comprising:

According to the present invention, a new cell culture device useful for analysis of function at a single cell level, or analysis of cell-to-cell communication, and a cell culture method can be provided.

It is a brief explanation drawing of a cell culture device of one embodiment of the first invention.

It is a brief cross-section view of the lower basal plate and the upper basal plate of the cell culture device of.

It is an explanation drawing of the cell culture method using the cell culture device of.

It is a brief explanation drawing of a cell culture device of one embodiment of the second invention.

It is a brief cross-section view of the lower basal plate and the upper basal plate of the cell culture device of

It is an explanation drawing of the cell culture method using the cell culture device of.

It is a photograph of a cell culture device produced in the Example, and (a) shows the upper basal plate, and (b) shows the lower basal plate.

It is a result of cell seeding test using the cell culture device produced in the Example, and it is a figure showing “the number of wells” based on “the number of cells per well” (for example, the number of wells in which one cell was introduced).

The cell culture device of the present invention is characterized by comprising a lower basal plate comprising a cell culture part having a bottom membrane, and an upper basal plate arranged on the upper side of the lower basal plate, and comprising a cell housing part having a bottom membrane in which a micropore are formed. When arranging the upper basal plate on the lower basal plate, the bottom membrane of the lower basal plate and the bottom membrane of the upper basal plate are arranged spaced apart up and down by a predetermined distance. The cell culture device of the present invention is not limited to those wherein the basal plates are arranged in two layers one above the other, and can be one wherein three or more layers are stacked.

The cell culture device of the present invention is useful for analysis of function at a single cell level, or analysis of cell-to-cell communication, etc.

Cells being the subject of culture of the cell culture device of the present invention are not particularly limited, and can be appropriately selected according to the purpose. For example, cells forming tissues such as skin, retina, cardiac muscle, blood vessel, nerve, organs, etc. collected from human body or animals; cell lines established from them; stem cells such as, mesenchymal stem cells (MSC), induced pluripotent stem cells (iPS cells), embryo stem cells (ES cells); cells forming tissues such as nerve, skin, cardiac muscle, liver, etc., induced to differentiate from stem cells can be exemplified.

In the following, the cell culture device of the first invention is explained.

The cell culture device of the first invention is characterized by comprising a lower basal plate comprising a cell culture part having a bottom membrane, and an upper basal plate arranged on the upper side of the lower basal plate, and comprising a cell housing part having a bottom membrane in which one micropore is formed, and when arranging the upper basal plate on the lower basal plate, the bottom membrane of the lower basal plate and the bottom membrane of the upper basal plate are arranged spaced apart up and down by a predetermined distance.

The cell culture device of the first invention can seed a single cell into the cell culture part of the lower basal plate at a high probability by allowing a cell to drop through one micropore formed in the bottom membrane of the cell housing part of the upper basal plate. Specifically, as compared to when conventionally seeding directly cells using a micropipette, it is possible to introduce only one cell into one cell culture part (well) at a high probability. Further, even when the surface area of the cell culture part is large, it is possible to introduce one cell at a high probability, and it can be suitably used for culture of adherent cells that require a large culture surface area, such as neurons.

In the following, each member of the cell culture device of the first invention is explained.

The lower basal plate is a plate comprising the cell culture part, and as material, for example, silicon wafer, glass wafer, and ceramic wafer such as quartz wafer can be exemplified. The thickness is preferably 200 μm or more, more preferably 200 to 500 μm, and further preferably 250 to 350 μm.

The cell culture part is a portion where the cells are seeded and cultured, and for example, it is a well (depressed area) formed in the lower basal plate. The shape of the inner space of the cell culture part is not particularly limited, and for example, shapes such as cylindrical shape, rectangular column shape, inverted truncated cone shape, inverted truncated pyramid shape, etc. can be exemplified. Inverted truncated pyramid shape is preferable, and inverted truncated quadrangular shape (inverted pyramid shape) is particularly preferable. One or more cell culture parts can be provided, and generally, they have a number corresponding to the number of cell housing parts of the upper basal plate, described in the following, and they are arranged in a position corresponding to the cell housing parts of the upper basal plate. The cell culture parts are preferably arranged in a large number in a grid pattern (array pattern).

In case the cell culture parts are arranged in a grid pattern, the cell culture parts are separated and comparted by a grid-patterned partitioning member (lower partitioning member) that constitutes the side walls (peripheral walls) of the cell culture parts. It is preferable that the concave portion formed on the lower part of the upper partitioning member of the cell housing parts of the upper basal plate described in the following is mated to all or a part of the upper part of the lower partitioning member, to perform position adjustment of the cell culture parts of the lower basal plate and the cell housing parts of the upper basal plate.

The size (diameter (diagonal line in case of polygon)) of the bottom membrane (cell culture membrane) of the cell culture part is preferably 100 to 1000 μm, more preferably 150 to 800 μm, and further preferably 200 to 600 μm.

Micropores may not be formed in the cell culture membrane, while it is preferable that a plurality of micropores are formed. Thereby, for example, in case cells are loaded on one of the surfaces of the cell culture membrane, it is possible to supply nutrient of the medium through micropores from the other surface. Further, in case cells are loaded on both surfaces of the cell culture membrane, it is possible to smoothly perform cell-to-cell communication via micropores.

The shape of micropores can be appropriately set according to types of cells or purpose of the culture, and for example, shapes such as circular shape, polygonal shape can be exemplified. The diameter of the micropores (diagonal line in case of polygon) is preferably a size that cells cannot pass through, preferably 20 μm or less, more preferably 0.1 to 10 μm, further preferably 0.5 to 5 μm, and particularly preferably 1 to 4 μm. The area ratio of micropores on the bottom membrane of the cell culture part depends on the diameter of the micropores, but is preferably 10 to 70%, more preferably 10 to 50%, and further preferably 10 to 20%. If it is within this range, supply of nutrient through micropores, or cell-to-cell communication can be smoothly performed, and in case it is a transparent material, high transparency can be maintained.

The membrane thickness of the cell culture membrane is preferably, for example 0.1 to 5 μm, more preferably 0.5 to 1.5 μm. If it is within this range, the mechanical strength as cell scaffold can be maintained, and in case it is a transparent material, high transparency can be maintained.

The material of the cell culture membrane is not particularly limited as long as it can be a cell scaffold, while from the viewpoint that cell observation is facile, it is preferable to be composed of transparent material. As transparent material, it can be an organic material, while inorganic material is preferable. Inorganic materials include, for example, those that are transparent, among nitride, oxide, oxynitride, etc. of silicon, titanium, zinc, tin, and aluminum. Specifically, silicon nitride (SiN), silicon oxide (SiO), titanium oxide (TiO), zinc oxide (ZnO), tin oxide (SnO), silicon oxynitride (SiON), titanium oxynitride (TiON), indium oxide (InO), indium tin oxide (ITO), etc. can be exemplified. Silicon nitride, silicon oxide, silicon oxynitride are preferable, and silicon nitride or silicon oxide is particularly preferable. These transparent inorganic materials can be used in combination of two or more. Further, a laminated membrane consisting of two or more types can be formed. For example, it can be an inorganic material with a transparent organic material coated on its surface.

Here, in the present specification, “transparent” means that the light transmittance at a wavelength of 500 nm to 600 nm is 70% or more, preferably 80% or more, and more preferably 90% or more.

It is preferable that the surface of the bottom membrane (cell culture membrane) of the cell culture part is modified with functionalized molecules, to promote cell adhesion or cell proliferation. Functionalized molecules are not particularly limited as long as they can be used for cell culture, and they include, for example, extracellular matrix such as collagen, proteoglycan, heparan sulfate proteoglycan, fibronectin, laminin, entactin, elastin, hyaluronic acid, tenascin, etc., cell adhesion peptide such as arginine-glycine-asparagine acid, leucine-asparagine acid-valine, arginine-glutamic acid-asparagine acid-valine, etc., or synthetic molecules such as poly-L-lysine, poly-L-ornithine, etc.

It is preferable that the lower basal plate comprises a supporting frame part (holder) supporting the lower basal plate. Thereby, when arranging the upper basal plate on the lower basal plate, it can be arranged with more stability. Further, since it can be used as a grasping part, the operability can be enhanced. As material of the supporting frame part, synthetic resin can be exemplified.

The upper basal plate is a plate arranged on the upper side of the above-mentioned lower basal plate, and comprising a cell housing part having a bottom membrane in which one micropore is formed. As material of the basal plate, a similar material as the lower basal plate can be used, and it can be the same material as the lower basal plate, or can be a different material. The thickness of the upper basal plate is preferably 200 μm or more, more preferably 200 to 500 μm, and further preferably 250 to 350 μm.

The cell housing part is a portion that can house cells, and is for example a well (depressed area) formed in the upper basal plate. The shape of the inner space of the cell housing part is not particularly limited, and for example, shape such as cylindrical shape, rectangular column shape, inverted truncated cone shape, inverted truncated pyramid shape, etc. can be exemplified. Inverted truncated pyramid shape is preferable, and inverted truncated quadrangular shape (inverted pyramid shape) is particularly preferable. One or more cell housing parts can be provided, and generally, they have a number corresponding to the number of cell culture parts of the lower basal plate, described in the above, and they are arranged in a position corresponding to the cell culture parts of the lower basal plate. The cell housing parts are preferably arranged in a large number in a grid pattern (array pattern). In case two or more cell housing parts are provided, each cell housing part has preferably the same constitution, so that cells drop in a uniform manner.

In case the cell housing parts are arranged in a grid pattern, the cell housing parts are separated and comparted by a grid-patterned partitioning member (upper partitioning member) that constitutes the side walls (peripheral walls) of the cell housing parts. As stated above, it is preferable to form a concave part in the lower part of the upper partitioning member, to mate the convex part of the upper part of the lower partitioning member of the cell culture part of the lower basal plate, to perform position adjustment of the two parts. Generally, when arranging the upper basal plate on the lower basal plate, a part of the cell housing part of the upper basal plate is introduced in the cell culture part of the lower basal plate. Therefore, it is preferred that the bottom membrane of the cell housing part of the upper basal plate is smaller than the bottom membrane of the cell culture part of the lower basal plate, for example about ⅓ to ⅔.

In the bottom membrane of the cell housing part, one micropore having a size large enough for a cell to pass through is formed. The one micropore is preferably formed in the center of the bottom membrane of the cell housing part. Thereby, when arranging the upper basal plate on the upper side of the lower basal plate, the micropore is positioned in the center upper side of the bottom membrane of the cell culture part of the lower basal plate, and it is possible to let a cell surely drop into the cell culture part of the lower basal plate. The shape of micropores can be appropriately set according to types of cells, and for example, shapes such as circular shape, polygonal shape can be exemplified. The diameter of the micropores (diagonal line in case of polygon) can be appropriately set according to the types of the cells, and it is preferably larger than a diameter of one cell and smaller than the total of diameters of two cells. For example, it is preferably a diameter of 3 to 50 μm, more preferably 8 to 20 μm. Specifically, for example, in case of neuron, since the diameter is about 8 μm, a micropore having a diameter of 10 to 15 μm is preferable. Thereby, it is possible to seed only one cell into one cell culture part, at high probability.

It is preferable that the upper basal plate comprises a supporting frame part (holder) supporting the upper basal plate. Thereby, when arranging the upper basal plate on the lower basal plate, it can be arranged with more stability. Further, since it can be used as a grasping part, the operability can be enhanced. As material of the peripheral wall, synthetic resin can be exemplified.

In the cell culture device of the first invention, when arranging the upper basal plate on the lower basal plate, the bottom membrane of the lower basal plate and the bottom membrane of the upper basal plate are arranged spaced apart up and down by a predetermined distance, and one micropore of the bottom membrane of the upper basal plate is arranged so that it is positioned in the upper side of the bottom membrane of the lower basal plate. At that time, it is preferable that the central parts of the bottom membranes are arranged to coincide with each other. Thereby, it is possible to let cells drop to the bottom membrane of the cell culture part of the lower basal plate reliably and accurately. The distance between the bottom membranes is preferably 5 to 500 μm, more preferably 20 to 250 μm, and further preferably 50 to 200 μm.

The embodiment of position adjustment of the lower basal plate and the upper basal plate is not particularly limited, and its examples include embodiment of mating concave and convex formed on each of the lower basal plate and the upper basal plate; embodiment of adjoining and mating each of the peripheral convex parts having large and small diameters (diagonal line in case of polygon) formed on the outer periphery of each of the lower basal plate and the upper basal plate; embodiment of placing the upper basal plate within the peripheral frame of the lower basal plate; embodiment of aligning the marks added to each of the lower basal plate and the upper basal plate and fixing them with a fixing tool; and embodiment of fixing by inserting the insertion rods to the insertion holes formed in each of the lower basal plate and upper basal plate, etc.

Specifically, as embodiment of mating concave and convex formed on each of the lower basal plate and upper basal plate, for example, its examples include embodiment wherein the lower basal plate comprises a convex part or a concave part, and the upper basal plate comprises a concave part or a convex part, the convex part or the concave part of the lower basal plate and the concave part or the convex part of the upper basal plate are mated to perform position adjustment of the cell culture part of the lower basal plate and the cell housing part of the upper basal plate. More specifically, an embodiment of mating the peripheral concave part or the peripheral convex part surrounding the upper basal plate to the peripheral convex part or the peripheral concave part surrounding the lower basal plate; or an embodiment where the upper part of the lower partitioning member separating a plurality of the grid-patterned cell culture parts of the lower basal plate constitutes the concave part, and a concave part corresponding to the upper convex part of the lower partitioning member is formed on the lower part of the upper partitioning member separating a plurality of cell housing parts of the upper basal plate corresponding to the lower partitioning member, to mate the grid-patterned convex part of the lower basal plate and the grid-patterned concave part of the upper basal plate can be exemplified. One convex part or concave part can be provided on each of the lower basal plate and the upper basal plate, or plural parts can be provided.

As embodiment of adjoining and mating the peripheral convex parts formed on each of the lower basal plate and the upper basal plate, its examples include an embodiment of arranging by adjoining and mating the peripheral convex part surrounding the periphery of the lower basal plate, and the peripheral convex part surrounding the upper basal plate, with a diameter (diagonal line in case of polygon) being one size larger or smaller than the convex part of the lower basal plate.

In case of such embodiment of position adjustment of the lower basal plate and the upper basal plate, the position adjustment of the cell culture part of the lower basal plate and the cell housing part of the upper basal plate in horizontal direction and in vertical direction (distance between the cell culture membrane of the upper basal plate and the cell culture membrane of the lower basal plate) can be easily and accurately performed, and the operability when culturing is enhanced.

Next, the production method of the cell culture device of the first invention is explained.